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Abstract ［Objectives］ This study was conducted to clarify the evolution characteristics of foxtail millet varieties in different ages and provide a basis for the breeding of new varieties.
［Methods］ A field experiment was carried out on 20 main foxtail millet varieties promoted in North China developed from the 1980s to 2000s. The physiological and biochemical indexes of different foxtail millet varieties in four ages were compared, including chlorophyll content, soluble protein content, glutamine synthetase (GS) activity and glutamate synthase (GOGAT) activity, and the correlation between enzyme activity and yield was analyzed.
［Results］ The chlorophyll SPAD values of the flag leaf and functional leaves of foxtail millet varieties decreased with the filling process. The SPAD values of the flag leaf, top second leaf and top third leaf were higher in the varieties developed in the 1990s and 2000s than those in the 1980s and 2010s. The activity of glutamine synthetase (GS) and glutamate synthase (GOGAT) showed a singlepeak curve in different foxtail millet varieties developed in the recent 30 years, and the peaks of the two were at 7 d and at 7 or 14 d, respectively. The activity of GS and GOGAT increased with the breeding age. In the period from 7 d after anthesis to the mature period, the decreases in the soluble protein content followed an order of 2010s, 2000s, 1990s and 1980s from small to large, indicating that the degradation rate of various enzyme sources and metabolic regulators in foxtail millet plants decreased during the improvement process. At 35 d after anthesis, the correlation coefficient between GS activity and yield was -0.247, that is, there was a negative correlation with yield. And there was a significant positive correlation between GOGAT activity and yield, and the correlation coefficient was as high as 0.455 at 7 d after anthesis.
［Conclusions］ Changes in the GS activity, GOGAT activity and soluble protein content in the flag leaf of foxtail millet varieties developed in recent years have a certain impact on yield.
Key words North China; Foxtail millet; Glutamine synthetase; Glutamate synthase; Age
Received: July 21, 2019Accepted: October 8, 2019
Supported by The Earmarked Fund for Modern Agroindustry Technology Research System (CARS0613.5A19); Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences (GXGC2018D02); Shandong Key R&D Program (2018GNC113016). Qinghua KONG (1995-), female, P. R. China, master, devoted to research on plant physiology.
*Corresponding author. Email: yguan65@163.com.
Foxtail millet ［Setaria italica (L.) Beauv.］ belonging to Setaria of Gramineae has a long history of cultivation and a wide range of planting. It is mainly distributed in arid and semiarid regions of northern China, and has high drought resistance, good tolerance to poor soil, high water use efficiency, rich nutrition and balanced composition［1-4］. Nitrogen is the essential and most demanding mineral nutrient in plant growth and development, and is associated with various life activities in plants. When nitrogen is sufficient, photosynthesis products can be rapidly transformed to synthesize nucleic acids, enzymes, proteins and chlorophyll to provide a material basis for plant life activities［5］. Higher plants cannot directly use nitrogen and can only absorb the combined nitrogen. The main nitrogen sources are inorganic nitrogen sources such as ammonium salts and nitrates, which enter nitrogen assimilation through glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle, and are finally converted into amino acids which are absorbed by plants［6-7］.
Nitrogen application is one of the important ways to increase yield during crop cultivation. However, the utilization efficiency of nitrogen fertilizer in China is low and the amount of nitrogen fertilizer is large, which not only increases production costs, but also seriously pollutes the environment and hinders the sustainable development of agriculture. Exploring the potential of efficient nitrogen utilization in crops and selecting varieties with high nitrogen use efficiency has become an important means to improve the efficient use of nitrogen in crops. Studies have shown that wheat, corn, rice and other crops have large differences in genotypes in nitrogen uptake and utilization［8-11］. The study of Chen et al.［12］ on foxtail millet showed that different varieties were significantly different in nitrogen use efficiency at seedling stage.
Nitrogen in plants is mainly in the form of soluble proteins, which include various enzyme sources and metabolic regulators. The change of soluble protein content in plant leaves is an important feature of leaf senescence, and the content can reflect the level of nitrogen metabolism in plants［13-14］. Han et al.［15］ showed that the soluble protein content in wheat flag leaf increased during grain filling stage, which was beneficial to prolong the photosynthetic function period of wheat flag leaf, thus increasing the accumulation of carbon and nitrogen compounds in grains. High assimilation of nitrogen refers to the capacity of plants to directly assimilate NH+4 in soil to organic nitrogen. With high activity of glutamine enzyme, crops can absorb NH+4 from soil quickly and store it in cytoplasm and vacuoles in large quantities to reduce nitrogen loss, thereby enhancing nitrogen metabolism and improving nitrogen utilization rate［16］. NH+4 in plant leaves can be produced by photorespiration or by reduction of NO-2, and the former produces NH+4 5-10 times more than the NH+4 produced by reduction［17］. Glutamine synthetase is capable of storing exogenous NH+4 in the amide group of glutamine to provide ammonia for the synthesis of other nitrogencontaining compounds in plants［18］. Glutamate synthetase (GOGAT) reduces the amine group of glutamine under the action of reduced ferredoxin or NADH to form two molecules of glutamic acid. Numerous studies have shown that GS activity in plant kernels and GS activity in leaves have a significant or extremely significant relationship with the formation of protein final content and protein synthesis［19-23］. Wang et al.［24］ showed that the GOGAT activity of wheat grain and the GS activity of the flag leaf showed a significant or extremely significant positive correlation with the protein content of wheat grain. Chen et al.［25］ found that the GOGAT activity in rice was significantly correlated with the leaf color values of the top first and top second leaves in canopy leaves in the study of the relationship between leaf color change and the activity of key enzymes in nitrogen assimilation under excessive nitrogen application. In this study, the 20 main foxtail millet varieties in North China from the 1980s up to now were used to analyze the changes of nitrogen metabolism related enzyme activity and soluble protein content in the flag leaf from flowering to maturity, aiming to clarify the evolution characteristics of foxtail millet varieties developed in different ages and the importance of GS and GOGAT enzyme activity on yield formation. This study will provide a theoretical basis for the breeding of varieties with high nitrogen use efficiency in North China summer foxtail millet areas.
Materials and Methods
Experiment materials
The experiment materials were 20 foxtail millet varieties that had been extensively promoted in the North China summer foxtail millet areas since the 1980s. The examination years and the breeding units are shown in Table 1. According to the breeding years of the tested foxtail millet varieties, the 20 foxtail millet varieties were divided into four periods, and the foxtail millet varieties in each period are shown in Table 2.
Experiment design
The experiment was carried out in 2016 at the Jinan Experimental Base of Institute of Crop Sciences, Shandong Academy of Agricultural Sciences. The soil was loam, and the topsoil was from 0 to 20 cm, which contained 16.9 g/kg of organic matter, 72.4 mg/kg of alkalihydrolyzable nitrogen, 18.8 mg/kg of available phosphorus, and 168.0 mg/kg of available potassium, and had a pH value of 7.8. As to fertilization, 375 kg of NPK (151515) ternary compound fertilizer and 750 kg of decomposed organic fertilizer were applied per hectare. The previous crop was winter wheat, and the cultivation and management were the same as the highyielding fields.
The experiment adopted randomized block design with three replicates. Each variety was one treatment (plot). Sowing was performed on June 26, followed by thinning at 4leaf stage and final singling at 5-6leaf stage, and the final density was 600 000 plants/hm2. The plants were harvested on September 30.
Physiological index determination and methods
Sampling: Five plants with similar growth state were selected from each variety at 5, 7, 21, 28 and 35 d after the flowering stage, and the flag leaf were cut, frozen in liquid nitrogen, and stored in a -40 ℃ refrigerator for the determination of enzyme activity.
Chlorophyll SPAD value determination
The chlorophyll contents of flag leaf, top second leaf and top third leaf were determined one week after anthesis with an SPAD502 chlorophyll content analyzer. Five plants were tested for each replicate, and each leaf was measured at 3 points, to obtain an average. The measurement was performed once every other week. Determination of soluble protein content in flag leaf
Referring to Handbook of Plant Physiology Experiment edited by Xiong［26］, the protein content of foxtail millet leaves was determined by Coomassie brilliant blue method.
Determination of glutamine synthetase and glutamate synthase activity in flag leaf
Enzyme solution extraction was performed according to the extraction method of soluble proteins. The enzyme activity was determined referring to Modern Plant Physiology Experiment edited by Tang［27］ and the method of Zhao et al.［28］.
Data processing
The data were statistically analyzed and plotted using Microsoft Excel 2007 and SAS statistical analysis software.
Results and Analysis
Variation trend of chlorophyll SPAD value
The chlorophyll SPAD values of the top three leaves of 20 foxtail millet varieties were determined. The results showed that the chlorophyll SPAD values of the flag leaf, top second leaf and top third leaf of foxtail millet varieties all had a downward trend with the filling process (Fig. 1-Fig. 3). In the varieties developed in the 1980s to the 2010s, the average SPAD values of the flag leaf were 51.8, 54.7, 54.1 and 52.5, respectively; the average SPAD values of the top second leaf were 52.8, 55.3, 54.1 and 53.3, respectively; and the average SPAD values of the top third leaf were 53.2, 55.5, 54.4 and 53.0, respectively. The SPAD values of the flag leaf, the top second leaf and the top third leaf were all higher in the varieties developed in the 1990s and 2000s, and lower in the varieties developed in the 1980s and 2010s. With the advancement of the breeding age, the trend of chlorophyll contents in different developed varieties increased first and then decreased. Meanwhile, the average SPAD values of foxtail millet varieties in various age basically followed an order of top third leaf>top second leaf>flag leaf.
Qinghua KONG et al. Analysis of Nitrogen Metabolism Related Enzymes and Related Physiological Indexes of Main Foxtail Millet Varieties Developed in North China
Variation trend of soluble protein content
Soluble proteins in the flag leaf contain enzymes that participate in various metabolic activities. Fig. 4 shows that the soluble protein contents in the flag leaf of the foxtail millet varieties developed in the 1980s, 1990s and 2010s showed a decreasing trend with the filling process. The 2000s varieties showed a trend of increasing first and decreasing then, and the soluble protein content reached its maximum at 7 d after anthesis. The soluble protein content of the flag leaf decreased from 7 d after anthesis to maturity in the varieties developed in different ages. Specifically, the value of the varieties developed in the 2010s decreased by 34.16%, which was lower than 2000s（35.98%）, 1990s（37.78%）and 1980s（38.91%）, indicating that the differences in the soluble protein content in the flag leaf of the foxtail millet varieties developed in the 2010s from the foxtail millet varieties developed in other ages increased with the number of days after anthesis increasing, and the nitrogen nutrition of the flag leaf in the late filling stage was gradually improved in the foxtail millet varieties as time went on. Variation of GS activity in the flag leaf of foxtail millet varieties developed in different ages
Glutamine synthetase (GS) is a key enzyme in nitrogen metabolism［29］. Fig. 5 shows that the dynamic changes of GS activity in the flag leaf of the varieties developed in different ages were basically the same, and they all showed a trend of increasing first and then decreasing, exhibiting a maximum at 7 d after anthesis. The differences in the GS activity of the flag leaf between the foxtail millet varieties developed in different ages were the highest at 14 d after anthesis, and the GS activity showed an upward trend with the breeding age, i.e., it ranked as 2010s>2000s>1990s>1980s, indicating that the nitrogen metabolism capacity of the foxtail millet flag leaf was improved and the duration also got longer with the breeding age, which was beneficial to the absorption and transport of nitrogen and thereby gave rise to a higher protein content.
Variation of GOGAT activity in the flag leaf of foxtail millet varieties developed in different ages
It can be seen from Fig. 6 that the dynamic changes of GOGAT activity in the foxtail millet varieties developed in different years were basically the same as the GS activity, and they increased first and then decreased, exhibiting a maximum at 7 or 14 d after anthesis. The GOGAT activity dropped faster from 14 d after anthesis. The GOGAT activity in the flag leaf of different varieties showed obvious chronological changes. The enzyme activity of the foxtail millet varieties developed in the 2010s was higher overall, while that of the varieties developed in the 1980s was lower.
Correlation between enzyme activity and foxtail millet yield in each period after anthesis
Correlation analysis was performed between the yields of the 20 foxtail millet varieties and the enzyme activity in each period after anthesis. The results (Table 3 and Table 4) showed that the correlation between GS activity and yield in each period began to increase gradually after 21 d, and the negative correlation between GS activity and yield was the largest at 35 d after anthesis, indicating that GS activity had a greater effect on yield at this time, and the yield gradually increased with the decrease of GS activity.
From the correlation between GOGAT activity and yield in each period, the correlation coefficient of the two was the highest at 0.455 at 7 d after anthesis, and there was a significant positive correlation, indicating that GOGAT activity had a greater effect on yield at 7 d after anthesis, and the yield gradually increased with the increase of GOGAT activity. According to the correlation analysis between soluble protein content and yield in each period, the soluble protein content was negatively correlated with the yield at 14 d after anthesis, and the correlation coefficient was -0.268 at 21 d after anthesis, indicating that the soluble protein content had a certain effect on the yield at this time, and the yield gradually increased with the decrease of the soluble protein content.
Discussion and Conclusions
Nitrogen is one of the essential elements for crop growth and an important factor affecting yield. The main source of nitrogen in plants is the inorganic nitrogen in the soil: ammonium salts and nitrates. NH+4 in plants is mainly assimilated by the GSGOGAT cycle pathway, and the NH+4 assimilation products provide precursors for the synthesis of other nitrogencontaining organic compounds［30］. GS is a key enzyme in the metabolic pathway, and the improvement of its activity is beneficial to improve the efficiency of nitrogen metabolism. GOGAT cooperates with GS to participate in the process of ammonia assimilation, catalyzing glutamine into glutamic acid and further synthesizing other amino acids and proteins［31］. Dai et al.［33］ found that the soluble protein content and glutamine synthetase (GS) activity of wheat flag leaf were significantly improved during the improvement of varieties. Yin［16］ found that the flag leaf SPAD value, soluble protein content, and flag leaf GS activity in the late filling stage, were improved during the improvement process of wheat. The results of this study showed that the GS and GOGAT activity of the varieties developed in all ages showed an upward trend with the breeding age, indicating that the cultivar improvement improved the GS and GOGAT activity of the foxtail millet varieties. The correlation analysis between enzyme activity and foxtail millet yield in each period after anthesis showed that the GS and GOGAT activity in the flag leaf of the foxtail millet varieties developed in recent years were beneficial to the increase of foxtail millet yield.
Soluble proteins contain various enzyme sources in the process of plant metabolism［32］. Han et al.［15］ found that soluble proteins in the flag leaf in the filling stage plays an important role in the increase of the "library" as a kind of "source" production material. The increase of soluble protein content in the flag leaf in the grain filling stage is beneficial to maintain the growth of the flag leaf and prolong the photosynthetic function, thus laying a material foundation for the accumulation of carbon and nitrogen in grains. This study showed that from 7 d after anthesis to maturity, the decreases of soluble protein content in the foxtail millet varieties developed in different ages ranked as the 2010s, the 2000s, the 1990s and the 1980s from small to large, indicating the degradation rates of various enzyme sources and metabolic regulators in the foxtail millet plants were reduced during cultivar improvement. The correlation analysis between soluble protein content and foxtail millet yield in each period after anthesis showed that changes of soluble protein content in the flag leaf of the foxtail millet varieties developed in recent years were beneficial to the increase of foxtail millet yield. The level of chlorophyll content in leaves is closely related to its photosynthetic capacity［33-34］. The results of this study indicated that the chlorophyll SPAD values of the upper three leaves of foxtail millet followed an order of the top third leaf, top second leaf and flag leaf from large to small, and the chlorophyll SPAD values in the upper three leaves were all the lowest in the foxtail millet varieties developed in the 1980s and the highest in the foxtail millet varieties developed in the 1990s. The chlorophyll SPAD values of the varieties developed after the 1990s showed a downward trend with the breeding age, indicating that the chlorophyll content increased first and then decreased.
In summary, in recent years, the nitrogen assimilation capacity of the foxtail millet varieties developed in recent years has been improved, which can provide a material basis for the formation of higher grain yield. Therefore, in the future breeding, we should pay attention to the breeding of varieties with high nitrogen use efficiency, so as to increase the yield of foxtail millet.
References
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［Methods］ A field experiment was carried out on 20 main foxtail millet varieties promoted in North China developed from the 1980s to 2000s. The physiological and biochemical indexes of different foxtail millet varieties in four ages were compared, including chlorophyll content, soluble protein content, glutamine synthetase (GS) activity and glutamate synthase (GOGAT) activity, and the correlation between enzyme activity and yield was analyzed.
［Results］ The chlorophyll SPAD values of the flag leaf and functional leaves of foxtail millet varieties decreased with the filling process. The SPAD values of the flag leaf, top second leaf and top third leaf were higher in the varieties developed in the 1990s and 2000s than those in the 1980s and 2010s. The activity of glutamine synthetase (GS) and glutamate synthase (GOGAT) showed a singlepeak curve in different foxtail millet varieties developed in the recent 30 years, and the peaks of the two were at 7 d and at 7 or 14 d, respectively. The activity of GS and GOGAT increased with the breeding age. In the period from 7 d after anthesis to the mature period, the decreases in the soluble protein content followed an order of 2010s, 2000s, 1990s and 1980s from small to large, indicating that the degradation rate of various enzyme sources and metabolic regulators in foxtail millet plants decreased during the improvement process. At 35 d after anthesis, the correlation coefficient between GS activity and yield was -0.247, that is, there was a negative correlation with yield. And there was a significant positive correlation between GOGAT activity and yield, and the correlation coefficient was as high as 0.455 at 7 d after anthesis.
［Conclusions］ Changes in the GS activity, GOGAT activity and soluble protein content in the flag leaf of foxtail millet varieties developed in recent years have a certain impact on yield.
Key words North China; Foxtail millet; Glutamine synthetase; Glutamate synthase; Age
Received: July 21, 2019Accepted: October 8, 2019
Supported by The Earmarked Fund for Modern Agroindustry Technology Research System (CARS0613.5A19); Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences (GXGC2018D02); Shandong Key R&D Program (2018GNC113016). Qinghua KONG (1995-), female, P. R. China, master, devoted to research on plant physiology.
*Corresponding author. Email: yguan65@163.com.
Foxtail millet ［Setaria italica (L.) Beauv.］ belonging to Setaria of Gramineae has a long history of cultivation and a wide range of planting. It is mainly distributed in arid and semiarid regions of northern China, and has high drought resistance, good tolerance to poor soil, high water use efficiency, rich nutrition and balanced composition［1-4］. Nitrogen is the essential and most demanding mineral nutrient in plant growth and development, and is associated with various life activities in plants. When nitrogen is sufficient, photosynthesis products can be rapidly transformed to synthesize nucleic acids, enzymes, proteins and chlorophyll to provide a material basis for plant life activities［5］. Higher plants cannot directly use nitrogen and can only absorb the combined nitrogen. The main nitrogen sources are inorganic nitrogen sources such as ammonium salts and nitrates, which enter nitrogen assimilation through glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle, and are finally converted into amino acids which are absorbed by plants［6-7］.
Nitrogen application is one of the important ways to increase yield during crop cultivation. However, the utilization efficiency of nitrogen fertilizer in China is low and the amount of nitrogen fertilizer is large, which not only increases production costs, but also seriously pollutes the environment and hinders the sustainable development of agriculture. Exploring the potential of efficient nitrogen utilization in crops and selecting varieties with high nitrogen use efficiency has become an important means to improve the efficient use of nitrogen in crops. Studies have shown that wheat, corn, rice and other crops have large differences in genotypes in nitrogen uptake and utilization［8-11］. The study of Chen et al.［12］ on foxtail millet showed that different varieties were significantly different in nitrogen use efficiency at seedling stage.
Nitrogen in plants is mainly in the form of soluble proteins, which include various enzyme sources and metabolic regulators. The change of soluble protein content in plant leaves is an important feature of leaf senescence, and the content can reflect the level of nitrogen metabolism in plants［13-14］. Han et al.［15］ showed that the soluble protein content in wheat flag leaf increased during grain filling stage, which was beneficial to prolong the photosynthetic function period of wheat flag leaf, thus increasing the accumulation of carbon and nitrogen compounds in grains. High assimilation of nitrogen refers to the capacity of plants to directly assimilate NH+4 in soil to organic nitrogen. With high activity of glutamine enzyme, crops can absorb NH+4 from soil quickly and store it in cytoplasm and vacuoles in large quantities to reduce nitrogen loss, thereby enhancing nitrogen metabolism and improving nitrogen utilization rate［16］. NH+4 in plant leaves can be produced by photorespiration or by reduction of NO-2, and the former produces NH+4 5-10 times more than the NH+4 produced by reduction［17］. Glutamine synthetase is capable of storing exogenous NH+4 in the amide group of glutamine to provide ammonia for the synthesis of other nitrogencontaining compounds in plants［18］. Glutamate synthetase (GOGAT) reduces the amine group of glutamine under the action of reduced ferredoxin or NADH to form two molecules of glutamic acid. Numerous studies have shown that GS activity in plant kernels and GS activity in leaves have a significant or extremely significant relationship with the formation of protein final content and protein synthesis［19-23］. Wang et al.［24］ showed that the GOGAT activity of wheat grain and the GS activity of the flag leaf showed a significant or extremely significant positive correlation with the protein content of wheat grain. Chen et al.［25］ found that the GOGAT activity in rice was significantly correlated with the leaf color values of the top first and top second leaves in canopy leaves in the study of the relationship between leaf color change and the activity of key enzymes in nitrogen assimilation under excessive nitrogen application. In this study, the 20 main foxtail millet varieties in North China from the 1980s up to now were used to analyze the changes of nitrogen metabolism related enzyme activity and soluble protein content in the flag leaf from flowering to maturity, aiming to clarify the evolution characteristics of foxtail millet varieties developed in different ages and the importance of GS and GOGAT enzyme activity on yield formation. This study will provide a theoretical basis for the breeding of varieties with high nitrogen use efficiency in North China summer foxtail millet areas.
Materials and Methods
Experiment materials
The experiment materials were 20 foxtail millet varieties that had been extensively promoted in the North China summer foxtail millet areas since the 1980s. The examination years and the breeding units are shown in Table 1. According to the breeding years of the tested foxtail millet varieties, the 20 foxtail millet varieties were divided into four periods, and the foxtail millet varieties in each period are shown in Table 2.
Experiment design
The experiment was carried out in 2016 at the Jinan Experimental Base of Institute of Crop Sciences, Shandong Academy of Agricultural Sciences. The soil was loam, and the topsoil was from 0 to 20 cm, which contained 16.9 g/kg of organic matter, 72.4 mg/kg of alkalihydrolyzable nitrogen, 18.8 mg/kg of available phosphorus, and 168.0 mg/kg of available potassium, and had a pH value of 7.8. As to fertilization, 375 kg of NPK (151515) ternary compound fertilizer and 750 kg of decomposed organic fertilizer were applied per hectare. The previous crop was winter wheat, and the cultivation and management were the same as the highyielding fields.
The experiment adopted randomized block design with three replicates. Each variety was one treatment (plot). Sowing was performed on June 26, followed by thinning at 4leaf stage and final singling at 5-6leaf stage, and the final density was 600 000 plants/hm2. The plants were harvested on September 30.
Physiological index determination and methods
Sampling: Five plants with similar growth state were selected from each variety at 5, 7, 21, 28 and 35 d after the flowering stage, and the flag leaf were cut, frozen in liquid nitrogen, and stored in a -40 ℃ refrigerator for the determination of enzyme activity.
Chlorophyll SPAD value determination
The chlorophyll contents of flag leaf, top second leaf and top third leaf were determined one week after anthesis with an SPAD502 chlorophyll content analyzer. Five plants were tested for each replicate, and each leaf was measured at 3 points, to obtain an average. The measurement was performed once every other week. Determination of soluble protein content in flag leaf
Referring to Handbook of Plant Physiology Experiment edited by Xiong［26］, the protein content of foxtail millet leaves was determined by Coomassie brilliant blue method.
Determination of glutamine synthetase and glutamate synthase activity in flag leaf
Enzyme solution extraction was performed according to the extraction method of soluble proteins. The enzyme activity was determined referring to Modern Plant Physiology Experiment edited by Tang［27］ and the method of Zhao et al.［28］.
Data processing
The data were statistically analyzed and plotted using Microsoft Excel 2007 and SAS statistical analysis software.
Results and Analysis
Variation trend of chlorophyll SPAD value
The chlorophyll SPAD values of the top three leaves of 20 foxtail millet varieties were determined. The results showed that the chlorophyll SPAD values of the flag leaf, top second leaf and top third leaf of foxtail millet varieties all had a downward trend with the filling process (Fig. 1-Fig. 3). In the varieties developed in the 1980s to the 2010s, the average SPAD values of the flag leaf were 51.8, 54.7, 54.1 and 52.5, respectively; the average SPAD values of the top second leaf were 52.8, 55.3, 54.1 and 53.3, respectively; and the average SPAD values of the top third leaf were 53.2, 55.5, 54.4 and 53.0, respectively. The SPAD values of the flag leaf, the top second leaf and the top third leaf were all higher in the varieties developed in the 1990s and 2000s, and lower in the varieties developed in the 1980s and 2010s. With the advancement of the breeding age, the trend of chlorophyll contents in different developed varieties increased first and then decreased. Meanwhile, the average SPAD values of foxtail millet varieties in various age basically followed an order of top third leaf>top second leaf>flag leaf.
Qinghua KONG et al. Analysis of Nitrogen Metabolism Related Enzymes and Related Physiological Indexes of Main Foxtail Millet Varieties Developed in North China
Variation trend of soluble protein content
Soluble proteins in the flag leaf contain enzymes that participate in various metabolic activities. Fig. 4 shows that the soluble protein contents in the flag leaf of the foxtail millet varieties developed in the 1980s, 1990s and 2010s showed a decreasing trend with the filling process. The 2000s varieties showed a trend of increasing first and decreasing then, and the soluble protein content reached its maximum at 7 d after anthesis. The soluble protein content of the flag leaf decreased from 7 d after anthesis to maturity in the varieties developed in different ages. Specifically, the value of the varieties developed in the 2010s decreased by 34.16%, which was lower than 2000s（35.98%）, 1990s（37.78%）and 1980s（38.91%）, indicating that the differences in the soluble protein content in the flag leaf of the foxtail millet varieties developed in the 2010s from the foxtail millet varieties developed in other ages increased with the number of days after anthesis increasing, and the nitrogen nutrition of the flag leaf in the late filling stage was gradually improved in the foxtail millet varieties as time went on. Variation of GS activity in the flag leaf of foxtail millet varieties developed in different ages
Glutamine synthetase (GS) is a key enzyme in nitrogen metabolism［29］. Fig. 5 shows that the dynamic changes of GS activity in the flag leaf of the varieties developed in different ages were basically the same, and they all showed a trend of increasing first and then decreasing, exhibiting a maximum at 7 d after anthesis. The differences in the GS activity of the flag leaf between the foxtail millet varieties developed in different ages were the highest at 14 d after anthesis, and the GS activity showed an upward trend with the breeding age, i.e., it ranked as 2010s>2000s>1990s>1980s, indicating that the nitrogen metabolism capacity of the foxtail millet flag leaf was improved and the duration also got longer with the breeding age, which was beneficial to the absorption and transport of nitrogen and thereby gave rise to a higher protein content.
Variation of GOGAT activity in the flag leaf of foxtail millet varieties developed in different ages
It can be seen from Fig. 6 that the dynamic changes of GOGAT activity in the foxtail millet varieties developed in different years were basically the same as the GS activity, and they increased first and then decreased, exhibiting a maximum at 7 or 14 d after anthesis. The GOGAT activity dropped faster from 14 d after anthesis. The GOGAT activity in the flag leaf of different varieties showed obvious chronological changes. The enzyme activity of the foxtail millet varieties developed in the 2010s was higher overall, while that of the varieties developed in the 1980s was lower.
Correlation between enzyme activity and foxtail millet yield in each period after anthesis
Correlation analysis was performed between the yields of the 20 foxtail millet varieties and the enzyme activity in each period after anthesis. The results (Table 3 and Table 4) showed that the correlation between GS activity and yield in each period began to increase gradually after 21 d, and the negative correlation between GS activity and yield was the largest at 35 d after anthesis, indicating that GS activity had a greater effect on yield at this time, and the yield gradually increased with the decrease of GS activity.
From the correlation between GOGAT activity and yield in each period, the correlation coefficient of the two was the highest at 0.455 at 7 d after anthesis, and there was a significant positive correlation, indicating that GOGAT activity had a greater effect on yield at 7 d after anthesis, and the yield gradually increased with the increase of GOGAT activity. According to the correlation analysis between soluble protein content and yield in each period, the soluble protein content was negatively correlated with the yield at 14 d after anthesis, and the correlation coefficient was -0.268 at 21 d after anthesis, indicating that the soluble protein content had a certain effect on the yield at this time, and the yield gradually increased with the decrease of the soluble protein content.
Discussion and Conclusions
Nitrogen is one of the essential elements for crop growth and an important factor affecting yield. The main source of nitrogen in plants is the inorganic nitrogen in the soil: ammonium salts and nitrates. NH+4 in plants is mainly assimilated by the GSGOGAT cycle pathway, and the NH+4 assimilation products provide precursors for the synthesis of other nitrogencontaining organic compounds［30］. GS is a key enzyme in the metabolic pathway, and the improvement of its activity is beneficial to improve the efficiency of nitrogen metabolism. GOGAT cooperates with GS to participate in the process of ammonia assimilation, catalyzing glutamine into glutamic acid and further synthesizing other amino acids and proteins［31］. Dai et al.［33］ found that the soluble protein content and glutamine synthetase (GS) activity of wheat flag leaf were significantly improved during the improvement of varieties. Yin［16］ found that the flag leaf SPAD value, soluble protein content, and flag leaf GS activity in the late filling stage, were improved during the improvement process of wheat. The results of this study showed that the GS and GOGAT activity of the varieties developed in all ages showed an upward trend with the breeding age, indicating that the cultivar improvement improved the GS and GOGAT activity of the foxtail millet varieties. The correlation analysis between enzyme activity and foxtail millet yield in each period after anthesis showed that the GS and GOGAT activity in the flag leaf of the foxtail millet varieties developed in recent years were beneficial to the increase of foxtail millet yield.
Soluble proteins contain various enzyme sources in the process of plant metabolism［32］. Han et al.［15］ found that soluble proteins in the flag leaf in the filling stage plays an important role in the increase of the "library" as a kind of "source" production material. The increase of soluble protein content in the flag leaf in the grain filling stage is beneficial to maintain the growth of the flag leaf and prolong the photosynthetic function, thus laying a material foundation for the accumulation of carbon and nitrogen in grains. This study showed that from 7 d after anthesis to maturity, the decreases of soluble protein content in the foxtail millet varieties developed in different ages ranked as the 2010s, the 2000s, the 1990s and the 1980s from small to large, indicating the degradation rates of various enzyme sources and metabolic regulators in the foxtail millet plants were reduced during cultivar improvement. The correlation analysis between soluble protein content and foxtail millet yield in each period after anthesis showed that changes of soluble protein content in the flag leaf of the foxtail millet varieties developed in recent years were beneficial to the increase of foxtail millet yield. The level of chlorophyll content in leaves is closely related to its photosynthetic capacity［33-34］. The results of this study indicated that the chlorophyll SPAD values of the upper three leaves of foxtail millet followed an order of the top third leaf, top second leaf and flag leaf from large to small, and the chlorophyll SPAD values in the upper three leaves were all the lowest in the foxtail millet varieties developed in the 1980s and the highest in the foxtail millet varieties developed in the 1990s. The chlorophyll SPAD values of the varieties developed after the 1990s showed a downward trend with the breeding age, indicating that the chlorophyll content increased first and then decreased.
In summary, in recent years, the nitrogen assimilation capacity of the foxtail millet varieties developed in recent years has been improved, which can provide a material basis for the formation of higher grain yield. Therefore, in the future breeding, we should pay attention to the breeding of varieties with high nitrogen use efficiency, so as to increase the yield of foxtail millet.
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